Synaptic transmission is highly dependent on the precise organization of proteins at the synapse. Therefore, understanding the mechanisms that govern this organization during development is a fundamental goal of developmental neuroscience. During the past few years the investigator has obtained compelling in vivo evidence that a family of proteins, termed Membrane Associated Guanylate Kinases (MAGUKs), plays fundamental roles in clustering ion channels and cell adhesion molecules at the synapse, using the fly neuromuscular junction as a model system. The central hypothesis of this application is that one of the regulatory mechanisms that controls the association of these proteins with the synaptic membrane is phosphorylation. The investigator has presented evidence that the synaptic localization of DLG, a fly MAGUK, is regulated by Ca2+/calmodulin dependent kinase II (CaMKII) activity and preliminary data suggests that this may result from DLG phosphorylation. The investigator therefore proposes to investigate this regulatory process which provides a mechanism by which the dynamic association of synaptic proteins with the synaptic apparatus may be controlled. In the first aim, the effects of altering CaMKII activity on the structure and physiology of synapses will be investigated. In addition, they will generate transgenic flies with mutations in dlg that prevent or mimic phosphorylation. In the second aim, they will use biochemical and molecular techniques, as well as studies in heterologous cells to define DLG phosphorylation sites, to determine if DLG and CaMKII interact directly in the intact animal, and to ascertain how DLG phosphorylation affects the association of DLG binding partners. Finally, in the third aim they will examine the significance of a different but related protein, CaMGUK, which is also thought to be involved in synapse organization. This characterization will include an examination of the anatomy and physiology of CaMGUK mutants, and the identification of interacting partners. They expect that the studies in this application will have a direct impact on how we view brain and neuromuscular disorders such as Alzheimer's disease and myasthenic syndromes, which are characterized by a progressive alteration of synaptic function, and provide valuable tools to design treatments for regeneration after injury.